血管紧张素Ⅱ对新生大鼠心肌成纤维细胞增殖及其信号转导机制的影响

Effect of AngiotensinⅡ on Myocardial Fibroblasts Proliferation and Their Signal Transduction Mechanism

目的研究血管紧张素Ⅱ(AngⅡ)对新生大鼠心肌成纤维细胞(MFs)增殖及蛋白激酶Cε(PKCε)和蛋白激酶Cα(PKCα)的表达及转位的影响,了解AngⅡ的促增殖和信号转导机制。方法以差速贴壁法原代分离提纯培养SD乳鼠MFs为实验模型,免疫组织化学法鉴定后传代培养,第2-4代MFs分为实验组(加AngⅡ10-6mol/L)和对照组(不加AngⅡ)培养;四氮唑盐(MTT)比色法检测MFs增殖;通过间接免疫荧光法观察实验组和对照组PKC亚型ε和α在细胞内的分布和定位,Image-Pro-Plus4.0版专业图像处理软件对荧光强度进行半定量统计。结果1.实验组MFs数量较对照组显著增加(P<0.001);2.实验组和对照组荧光显微镜下初步观察:对照组PKC亚型ε存在于细胞膜、胞质和核-细胞骨架3个部分,但均较弱;实验组PKC亚型ε在细胞膜、胞质和核-细胞骨架3个部分强度明显增强,尤以核-细胞骨架为甚;对照组PKC亚型α主要存在于核-细胞骨架;实验组PKC亚型α在细胞膜、胞质和核-细胞骨架3个部分强度均明显增加,尤以核-细胞骨架为甚;且PKC亚型ε和α荧光强度半定量比较,实验组均显著高于对照组(Pa<0.001)。结论AngⅡ有促进MFs增殖的作用,PKC亚型ε、α可能在MFs信号转导过程中起重要作用。

Objective To study the effect of angiotensin Ⅱ（AngⅡ） on myocardial fibroblasts（MFs） proliferation,the expression and transposition of protein kinase C epsilon（PKCε） and alpha（PKCα）,and to find out the mechanism of AngⅡpromoting proliferation and signal trarsduction.Methods The primary culture neonate rat＇s MFs was used depending on the different time of cell adherence,by the method of immunohistochemical method identifying MFs,2-4 generations MFs were divided into experimental group and control group,experimental group was added with AngⅡ 10-6 mol/L,and nothing was added to control group.Colorimetric method of metrazolium salt（MTT） was used to detect the MFs proliferation; indirect immunofluorescence was used to detect the distribution and location of PKCε and PKCα,then Image-Pro-Plus 4.0 was used to add up fluorescence intensity.Results 1.The number of MFs in experimental group increased much more than that in control group and there was obviously statistical significance（P〈0.001）.2.Under fluorescence microscope,the expression and transposition of PKCε and PKCα were observed.In experimental group,PKCε and PKCα resided in plasmalemma,endochylema and nucleo-cytoskeleton,compared with control group,fluorescence intensity obviously enhanced,especially in nucleo-cytoskeleton.In control group,PKCε and PKCα resided in nucleo-cytoskeleton.Under the effect of 10-6 mol/L AngⅡ,PKCε and PKCα resided in plasmalemma,endochylema and nucleo-cytoskeleton and fluorescence intensity were enhanced obviously,especially in nucleo-cytoskeleton;moreover,fluorescence intensity discrepancy had notable statistical significance between experimetal group and control group（Pa〈0.001）.Conclusions AngⅡcan induce the proliferation of MFs.PKCε and PKCα maybe play important roles in the signal transduction process of MFs.